Controlled Crystallization of Calcium Carbonate via Cooperation of

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Article Cite This: ACS Omega 2018, 3, 16681−16692

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Controlled Crystallization of Calcium Carbonate via Cooperation of Polyaspartic Acid and Polylysine Under Double-Diffusion Conditions in Agar Hydrogels Norio Wada,* Naohiro Horiuchi, Miho Nakamura, Kosuke Nozaki, Akiko Nagai, and Kimihiro Yamashita

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Department of Inorganic Biomaterials, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10, Kanda-Surugadai, Chiyoda, Tokyo 101-0062, Japan ABSTRACT: We demonstrated the crystallization of calcium carbonate in the presence of polyaspartic acid (PAsp) and polylysine (PLys) under double-diffusion conditions in agar hydrogels. The crystallization of precipitated CaCO3 was discussed based on the equivalency rule, and it was suggested that the nucleation of CaCO3 in the presence of PLys and PAsp occurred heterogeneously on the polypeptide assemblies that act as templates for nucleation. With PLys alone, the precipitates had calcite as the major component and vaterite as the minor component, where calcite and vaterite adopted dendritic and spherulitic morphologies, respectively. In the presence of PAsp alone and a mixture of PLys and PAsp, the precipitates comprised calcite spherulites only. The spherulites had a hierarchal structure with an inner part (the core) having a loose structure and an outer part composed of calcite crystals that grew radically from the core. The cores completely dissolved during crystallization, resulting in the formation of spherulites with a hollow structure. It was speculated that the core consists of polypeptide assemblies and poorly crystalline CaCO3. PAsp and PLys exerted synergistic effects on calcite spherulite formation, where the spherulite and hollow sizes could be controlled by changing the relative amount of PLys and PAsp.

1. INTRODUCTION Biomaterials obtained by biomineralization consist of inorganic−organic composites, where the main inorganic materials are calcium carbonate and calcium phosphate. Formation of biomaterials usually progresses at the interface between the inorganic component and the organic component with specific proteins. In biomineralization systems, it is well known that the organic matrix plays a key role in the polymorph selection and control of the morphology and crystal orientation.1−4 In general, it is suggested that organic matrix plays the following roles in biomineralization: (a) act as a site for heterogeneous nucleation and (b) inhibit the growth of the crystals via adsorption. Notably, the reactive functional groups, such as carboxyl groups, play important roles in biomineralization. The carboxylate groups provide nucleation sites for the inorganic component based on the arrangement of the carboxylate groups and can bind calcium ions and induce a local high concentration of Ca2+ ions in the vicinity of the carboxylate groups.1−10 There are still many unknowns related to how organic matrixes affect the crystallization process. This issue is still the subject of research and is generally studied through in vitro experiments mimicking biomineralization. Precipitation in biomineralization processes is mostly controlled by diffusion of the precipitating components to the site where precipitation is initiated (i.e., under convectionfree conditions). Agar hydrogels having a three-dimensional and porous structure contain a large amount of free water and © 2018 American Chemical Society

provide an amenable environment for performing biological functions. In addition, the gel is a transport medium where convection is suppressed. Hence, transfer of the reactant ions in a gel system is governed by volume mass diffusion without the convection phenomenon.11 Moreover, agar gel is chemically stable and does not interact with biomolecules and reactant ions.12,13 Hence, it is expected that agar matrixes do not directly affect CaCO3 crystallization. Therefore, agar gel systems are powerful tools for studying the effect of additives on CaCO3 crystallization as a model of biomineralization.13 Under physiological environment, CaCO3 may exist as three anhydrous crystalline polymorphs; calcite is the most thermodynamically stable form, aragonite is metastable, and vaterite is the least stable. The crystallization of CaCO3 has attracted the attention of many researchers owing to its importance in various fields, such as industrial and technological applications, environmental science, etc. Therefore, understanding the process of CaCO3 crystallization and the effects of additives, especially polypeptides, is still an important issue. It is well known that polyamino acids with carboxyl groups as polyanions control the crystallization of CaCO3 because the groups can interact with Ca2+ ions in a solution.14−19 On the other hand, polyamino acids with amino Received: September 20, 2018 Accepted: November 27, 2018 Published: December 5, 2018 16681

DOI: 10.1021/acsomega.8b02445 ACS Omega 2018, 3, 16681−16692

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the equivalent concentration shifted toward the Ca2+ source during diffusion over time, and simultaneously the equivalent concentration at the position increased. Hence, a shift in the first precipitation position indicates a change in the time when nucleation occurs. Accordingly, the crystallization behavior of CaCO3 can be discussed by comparing the waiting time and the first precipitation position obtained in the presence of additives with those obtained without the additives. 2.2. CaCO3 Precipitation by the Double-Diffusion Method. The experimental system for the gel method was described in our previous studies.25,26 Here, we briefly explain the experimental procedure. The crystallization of CaCO3 was studied by employing the double-diffusion method and utilizing three acrylic tube chambers containing a Ca2+ source, a gel column, and a CO32− source. These tubes were separated by a cellophane dialysis membrane. Solutions of 0.2 M Na2CO3 (8 mL) and 0.1 M CaCl2 (8 mL) were poured into the respective chambers located on both sides of the gel column (50 mm length; 20 mm diameter). The gel was prepared by dissolving 0.3 wt % of agarose in boiling deionized (Milli-Q) water (10 mL). The chemicals used, Na2CO3 and CaCl2 (Wako Pure Chem.), were of reagent grade, and the agarose was SeaKem LE Agarose (Lonza). Ultrapure water was produced using a Milli-Q high-purity water system. The pH values of the CaCl2 and Na2CO3 solutions were 6.2 and 11.2, respectively. Two different polypeptides (Alamana Polymers), PAsp (sodium salt, MW = 4200, about 20 equiv of acid groups per molecule) and PLys (hydrobromide, MW = 1400, about 10 equiv of basic groups per molecule), were prepared and various amounts (2 and 4 mg/10 mL for PLys and 1 and 2 mg/10 mL for PAsp) were added to the 0.3% agar solution before gelation. Under all experimental conditions, the pH of the gel solutions was adjusted to 8.0 using the Tris−HCl buffer so as to make the degrees of protonation of the amino groups of PLys and deprotonation of the carboxyl groups of PAsp to be similar when the polypeptides were added to a gel solution. These degrees are discussed in a later section. The experiments were run in a thermostatic chamber kept at 28 °C for three days. To discuss the nucleation of CaCO3 in the presence of PAsp and PLys, the waiting time was measured in increments of 0.5 h, and the first precipitation position, practically determined as the distance from the dialysis membrane of the CO32− chamber to the CO32− source side of the first visible precipitation disk, was measured in 0.2 mm increments using an optical microscope at 20× magnification. To estimate the functional dissociation state of the polypeptides and the dominant carbonate species in the precipitation regions, the pH of the agar hydrogel interior at the start, at the time when first visible precipitation occurred, and at the end of the experiments was checked using pH indicators (pH test papers) such as Cresol red (pH 7.2−8.8), Thymol blue (pH 8.0−9.6), and Alizarin yellow (pH 10.0−12.0). 2.3. Characterization of Precipitates. The portion of the gel column containing the CaCO3 precipitates was sliced at the end of the experiments. The gel matrix was removed from the precipitate in the slice by washing with hot distilled water and the precipitate was then dried at room temperature. Identification of the precipitated phase was performed by Xray powder diffraction (XRD) with Ni-filtered Cu Kα radiation (λ = 1.5406 Å) and Fourier transform infrared (FTIR) spectroscopy at 2 cm−1 resolution using the KBr pellet technique. Identification of the CaCO3 polymorph from the

groups as polycations are generally considered to be less active because they can weakly combine with Ca2+ ions through chelation.20−22 In nature, biomineralization proceeds in the coexistence of many kinds of polyamino peptides. However, many in vitro experiments on biomineralization are carried out with proteins comprised of a single polyamino peptide to facilitate analysis of its effects on the in vitro crystallization of biomaterials. Moreover, synergistic effects at play in the protein may be overlooked when studying the effects of an individual peptide in a protein on biomineralization. Thus, studying combinations of multiple amino acids leads to a better understanding of the roles of polypeptides in biomineralization. The effects of a mixture of polypeptides on the crystallization of CaCO3 are of particular importance in the development of strategies for their controlled synthesis of organic and inorganic composites using biomimetics. The overarching aim of this study is to provide useful insight into the effects of polypeptides on CaCO3 biomineralization for biomimetic syntheses of inorganic materials. Hence, we studied CaCO3 crystallization in the presence of two kinds of polypeptides, polyaspartic acid (PAsp) as an acidic and cationic polypeptide with carboxyl groups in its structure and polylysine (PLys) as a basic and anionic polypeptide with amino groups, using the double-diffusion method in agar hydrogels.

2. EXPERIMENTAL SECTION 2.1. Precipitation in Gel under Double-Diffusion Conditions. It was verified that nucleation in a gel under double-diffusion conditions occurs at the point when CA = CB = Cm, where CA and CB are the concentrations of both precipitating components (i.e., the nuclei of structure AB), and Cm is the minimum concentration (critical concentration) required for nucleation; in addition, the condition (Cm)2 > Ks has to be satisfied, where Ks is the solubility product.23,24 This criterion incorporates the dual conditions for nucleation and is termed the “equivalency rule”. According to the equivalency rule, the important parameters for understanding the nucleation behavior are the waiting time, defined as the time elapsed from the experiment onset to the first observation of visible precipitation, and the first precipitation position (where the first visible precipitate appears in gel). The first precipitation position is explained as the position for critical nuclei to nucleate. The waiting time comprises two following time periods: (1) the time needed for a reactant solution to move from undersaturation at the experiment onset to threshold supersaturation for the production of the critical nuclei and (2) the time needed for the nuclei to grow to an observable size. The time required for (1) is affected by the nucleation process (homogeneous or heterogeneous nucleation) and the time required for (2) reflects the degree of growth inhibition due to adsorbed additives.12 During crystallization in gels, volume mass-transfer occurs by diffusion, thus theoretical treatments are generally based on Fick’s second law of diffusion. Therefore, we previously used the computerized simulation to analyze the relationship between the reactant diffusion time and the position of equivalent concentration based on the equivalency rule and the algebra of the Gauss error function approximated by Abramowitz and Stegun for Fick’s second law.5,25 Under double-diffusion conditions with CaCl2 (0.1 M) and Na2CO3 (0.2 M) solutions, the simulation indicates that the position of 16682


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obtained FTIR spectra can be performed by utilizing the fact that the vibrational bands of the CO32− ion in the CaCO3 polymorphs appear at different positions. The presence of the gel matrix and polypeptides in the CaCO3 precipitates was analyzed using FTIR spectroscopy and micro-FTIR spectroscopy. For the acquisition of the FTIR spectra of PAsp and PLys, KBr bulk crystals were soaked in a small quantity of a solution of PAsp or PLys and then dried. The KBr bulk crystals were ground to prepare a KBr pellet. The morphology of the precipitated CaCO3 and the presence of the gel matrix and polypeptides entrapped on/in the precipitates were observed by using the secondary electron (SE) and backscattered electron (BSE) modes of a scanning electron microscope (SEM). The BSE mode is useful for differentiating the chemical composition of a sample because accumulations of elements with a higher atomic number appear lighter than those with a lower atomic number. A BSE micrograph shows compositional contrast in a sample. Hence, when organic matrixes having chemically similar composition are included in a sample, it is difficult to identify individually the presence of each of them in a sample. To examine the polypeptide and gel matrix within the precipitates in detail, the precipitates were immersed in 0.01 M HCl to remove the mineral component and studied by using FTIR microscopy.

Table 1. Average Values and the Standard Derivations of the First Precipitation Position and Increase in Waiting Time polypeptides control PLys PAsp PLys and PAsp PLys and PAsp PLys and PAsp PLys and PAsp

amount added (mg/10 mL)

first precipitation position (mm)a

increase in waiting time (h)b

± ± ± ±

0 4 2 4 and 2

25.0 23.8 23.2 23.4

0.3 0.2 0.2 0.2

0 3.0 ± 0.3 4.0 ± 0.3 5.5 ± 0.4

4 and 1

23.4 ± 0.2

5.0 ± 0.4

2 and 2

23.2 ± 0.2

5.5 ± 0.3

2 and 1

23.2 ± 0.2

4.5 ± 0.3

The first precipitation position is defined as the distance from the gel interface adjoining the CO32− chamber. bThe increase in waiting time is defined as a delay in time compared with control. The waiting time in control was 20 h. a

3. RESULTS AND DISCUSSION 3.1. Influence of Polypeptides on First Precipitation Position and Waiting Time. The photographs in Figure 1

Figure 1. Photographs of precipitation disks in the gel column: (a) precipitation disk in gel in the absence of polypeptides at the end of the experiment; (b) precipitation disk in gel in the presence of a mixture of PLys (2 mg) and PAsp (1 mg) at the end of the experiment; (c) first precipitation disk formed in the presence of a mixture of PLys (2 mg) and PAsp (1 mg) at a precipitation position of 23.2 mm and a waiting time of 24.5 h. The right- and left-hand sides of the gel column were in contact with Na2CO3 and CaCl2 source chambers, respectively. Red arrows mark the first precipitation position in (a) and (b). The scale bar is 1 cm.

Figure 2. SE mode SEM micrographs of CaCO3 precipitates obtained with PAsp alone, PLys alone, and without either of them (control): (a) without polypeptides; (b−e) with PLys alone (4 mg); and (f) with PAsp alone (2 mg). The scale bar is 20 μm.

position where nucleation occurs (Figure 1a,b). The first precipitation positions and the waiting times obtained in the presence and absence of the polypeptides are summarized in Table 1. The average values and the standard derivations of the first precipitation position and the waiting time were obtained from three measurements of the first precipitation position on each experimental condition, that is, n = 3, respectively. We already described in section of Introduction that carboxyl groups and amino groups can interact with Ca2+ ions. With regard to the influence of the interaction on change in Ca2+ ion concentration in a gel, we observed that a decrease in the concentration of Ca2+ ions due to the interaction between their groups and Ca2+ ions is neglected because small amounts of the polypeptides were added in the present experiment.5,25 It is

represent the CaCO3 precipitation disks at the end of the experiment and at first precipitation in the presence and absence of the polypeptide, respectively. The first precipitation of CaCO3 appeared perpendicular to the diffusion direction, and the disks formed with polypeptides became narrower than those formed without polypeptides (control). The crystallization of CaCO3 proceeded toward the chamber containing the Ca2+ ions during the experimental run. The position of the right boundary of the precipitation disk is the first precipitation 16683


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Figure 4. XRD pattern and FTIR spectra of the precipitates formed in the presence of PLys alone (4 mg): (a) XRD pattern of the precipitates and (b) FTIR spectra of PLys alone (solid green line) and CaCO3 precipitates (solid red line).

Figure 3. FTIR spectra of pure calcite (a), agar gel (b), residue of calcite crystals with HCl treatment (c), and calcite crystals formed in the control (d).

During the experimental run, the pH increased in the direction from the Ca2+ source side to the CO32− source side in the gel, indicating that the diffusion of the reactant induces the formation of a pH gradient in the gel. Under all experimental conditions, the pH at the time of first visible precipitation in the presence and absence of the polypeptides was in the range of 9.6−9.8. The width of the regions of CaCO3 precipitation ranged from ca. 5 to 10 mm at the end of the experiment. The precipitation widths depended on the amount and kind of polypeptides added. The pH of the region of CaCO 3 precipitation at the end of the experiment varied from 9.4 to 10.6 along the width of the precipitation zone. The pH measurements indicate that the dominant carbonate species at the position of first precipitation and in the regions of subsequent crystallization was CO32−. The evolution of the pH in this region was similar regardless of the amount and the kind of polypeptides added. The functional dissociation states of PLys and PAsp in the gel column during CaCO3 crystallization were estimated. At the start of the experiments at pH 8.0, the degree of protonation of the amino groups of PLys was estimated to be ca. 96%, as the pKa (the acidic dissociation constant) of PLys is ca. 9.4,27 whereas the degree of deprotonation of the carboxyl groups of PAsp (pKa = ca. 5.2)28 was ca. 100%. At the first precipitation position (pH 9.6−9.8), the degree of protonation of the amino groups of PLys was ca. 39% (pH = 9.6) and ca. 29% (pH = 9.8), whereas the carboxyl groups of

believed that the nucleation phenomenon in the control is homogeneous because the agar gel dose not interact with biomolecules and reactant ions, that is, the gel itself does not influence CaCO3 nucleation.13 The first precipitation position and the waiting time were dependent on the amount of PLys and PAsp added. In the presence of PLys and PAsp, the first precipitation position shifted toward the CO32− source chamber and the waiting time increased in comparison to the first precipitation position and waiting time for the control. On the basis of the discussion of nucleation in the doublediffusion method presented above, the shift in the first precipitation position toward the CO32− source chamber indicates that the nucleation of CaCO3 occurs at a lower CaCO3 supersaturation than that of the control, suggesting that a heterogeneous nucleation occurs in the presence of the polypeptide. The increase in the waiting time is attributed to the inhibition of the subsequent growth of the nuclei via the adsorption of the mobile polypeptides in the gel.4,25 3.2. Evolution of pH in Gel Column and Estimation the Functional Dissociation State of the Polypeptides and the Dominant Carbonate Species. In the experiments with and without polypeptides, the pH of the agar gels at the time of the first visible precipitation and at the end of the experiments was examined by using pH indicators. Before the experiments, the gel column had a homogeneous pH of 8.0. 16684


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Figure 5. SE and BSE mode high-magnification SEM micrographs of CaCO3 precipitates obtained with PAsp alone and PLys alone: (a−f) with PLys alone (4 mg) and (g−i) PAsp alone (2 mg). Note that panels, (a, b, d, e, g, and h) are the SEM micrographs acquired in the SE mode and panels, (c, f, and i) are the SEM micrographs in the BSE mode. The scale bars are 20 μm (a, d, g) and 5 μm (b, c, e, f, h, i).

XRD, SEM, and FTIR, respectively. Figure 2 shows the SEM micrographs of the CaCO3 precipitates obtained with PAsp alone, PLys alone, and without either of them (control). In the control, the precipitates adopted a rhombohedral morphology with a rough surface (Figure 2a), and the polymorph was identified as calcite from the characteristic rhombohedral morphology, which corresponds to the shape of the (10.4) plane of calcite. There are interesting studies that have discussed the incorporation of agar gel matrix within calcite crystals during growth.33,34 They described that the incorporation was controlled by the strength of a given gel increased with its solid content and the growth rate of the calcite crystals and also stated that a high-density gel (>0.5 wt %), and a high growth rate was incorporated into the calcite crystals, whereas a low-density gel (≤0.5 wt %) and a low growth rate led to the formation of almost gel-free composites. Hence, we examined the presence of the agar gel matrix within the calcite crystals precipitated without the polypeptides in the agar gel corresponding to a low-density gel. The FTIR spectra of the agar gel alone, calcite crystals formed in the control, and a residue of the calcite crystals with HCl treatment are shown in Figure 3. We cannot explain the difference in the spectrum between Figure 3b,c. However, from the weak broad bands at the region of 1050−1060 cm−1 in Figure 3c,d, we judge that a trace amount of the agar gel matrix is present in/on the calcite crystals. Hence, considering the surface topography of the calcite crystals formed in the control (Figure 2a), the gel matrixes inhibit a layer-by-layer growth of the calcite crystals through the phenomenon that the gel matrixes anchor to the crystals, resulting in the rough surface of calcite crystals. The SEM micrographs of the calcite crystals formed in the control acquired in SE and BSE mode did not allow to identify the presence of the gel matrix on the crystals. The FTIR spectrum of the precipitates showed the typical vibrational bands of

PAsp were almost completely dissociated. The addition of PAsp induces the formation of a PAsp−Ca2+ complex in the gel column containing Ca2+ ions, and the complex may act as a heterogeneous nucleation site for CaCO3 formation.19 On the other hand, the neutral amino groups of PLys can weakly combine with Ca2+ ions through chelation, with the possibility of forming PLys−Ca2+ complexes,20,21 suggesting that the PLys complex could act as a heterogeneous nucleation site for CaCO3 formation. Nonstoichiometric mixtures of oppositely charged polypeptides lead to the formation of polyion complexes through electrostatic interactions between the protonated amino (−NH 3 + ) groups and carboxylate (−COO−) groups; these polyion complexes then undergo assembly to form structural particles that are prepared by tuning the preparation conditions, such as the charge ratio of the anionic-to-cationic polymers and concentration.29 It is a well-established fact that polyion complex assemblies consist of a neutralized, hydrophobic core and a shell of excess polyion chain, which stabilizes the assemblies, that is, the absolute charge of the complex is that of the polyion in excess.29−31 In the CaCO3 precipitating regions with pH values between 9.4 and 10.6, the degree of protonation of the amino groups of PLys changes from ca. 50% (pH = 9.4) to ca. 10% (pH = 10.4), whereas the degree of deprotonation of the carboxyl groups of PAsp is 100%. Therefore, we believe that a mixture of PAsp and PLys in a gel may form polyion complex assemblies with a negative net charge and propose that the negatively charged sites of the polyion complex assemblies attract Ca2+ ions and provide heterogeneous nucleation sites for CaCO3 formation.32 3.3. Morphologies and Crystal Phases of Precipitated CaCO3. The polymorphs and morphologies of the resulting CaCO3 precipitates isolated from the gel matrixes and the presence of polypeptides in the precipitates were analyzed by 16685


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Figure 6. SE and BSE mode SEM micrographs of spherulites precipitated in the presence of a mixture of PLys and PAsp: (a−c) with PLys (4 mg) and PAsp (2 mg); (d−f) with PLys (4 mg) and PAsp (1 mg); (g−i) with PLys (2 mg) and PAsp (2 mg); and (j−l) with PLys (2 mg) and PAsp (1 mg). Note that panels, (a, b, d, e, g, h, j, and k) are the SEM micrographs acquired in the SE mode and panels, (c, f, i, and l) are the SEM micrographs in the BSE mode. The scale bars are 20 μm (a, d, g, j) and 2 μm (b, c, e, f, h, i, k, l).

calcite at 712 and 876 cm−1, supporting the fact that the crystal phase of the rhombohedra was calcite (Figure 3d).35 With PLys alone, the precipitates comprised of mainly distorted rhombohedra and dendrites with a star-like shape and a few spherulites. The XRD pattern of the precipitates in the presence of PLys alone indicated a major calcite component and a miner vaterite component (Figure 4a). The polymorphic ratio of calcite to vaterite in the precipitates was calculated from the integrated intensity of the XRD peaks of the (10.4) plane (θ = 29.4°) of calcite and the (11.2) plane (θ = 27.0°) of vaterite according to the following equation: y =1/(1 + 2.9Iv/Ic),26 where y is the calculated weight fraction of calcite and Iv and Ic are defined as the integrated intensity of the characteristic diffraction peaks of the (11.2) plane of vaterite and the (10.4) plane of calcite, respectively. The estimated fractions of calcite and vaterite were ca. 87% and ca. 13%, respectively. The FTIR spectrum obtained from the precipitates formed with PLys suggested that there was considerable overlapping between the broad bands of PLys and gel matrix at around 1060 cm−1 (Figures 3c and 4b). Hence, the presence of PLys in the precipitates was confirmed by its bands at around 1640 cm−1 (Figure 4b). The FTIR spectrum of the precipitates also revealed a strong band of calcite at 712 cm−1 and a weak band of vaterite at 744 cm−1

(Figure 4b).35 The relationship between the polymorphs and the crystal morphology was analyzed using FTIR spectroscopy. The FTIR spectrum of the precipitates with rhombohedral and dendritic shapes showed the typical vibrational bands of calcite at 712 and 875 cm−1 (date not shown), indicating that the rhombohedra and dendrites correspond to the calcite crystal phase.35 In contrast, the FTIR spectrum of the spherulites indicated the typical vibrational bands of vaterite at 744 and 876 cm−1 (date not shown), revealing that the crystal phase of the spherulites was vaterite.35 The SEM micrographs of the dendrites acquired in SE and BSE mode suggest that the vertices of the rhombohedral faces grew more rapidly (through two-dimensional nucleation) than the center, resulting in a star-like and dendritic morphology, and the dendrite surfaces were formed through parallel overgrowth by PLys adsorption (Figures 2b−d and 5a−c). The high-magnification SEM micrographs of the spherulites acquired in SE and BSE modes reveal that the spherulite consisted of thin platelet-like crystals approximately 0.05 μm thick (Figure 5e,f). The spaces between the platelet-like crystals were filled with organic matrixes (PLys and gel matrixes) (Figure 5d−f). Therefore, PLys as a positively charged additive promoted the formation of vaterite, which was the least stable phase and caused relatively major changes in the calcite morphology. 16686


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Figure 7. FTIR spectra and XRD pattern of the spherulites formed with PAsp alone (2 mg): (a) FTIR spectra of the spherulites (solid red line) and PAsp alone (solid orange line) and (b) XRD pattern of the spherulites.

In the presence of PAsp alone and a mixture of PLys and PAsp, a dramatic change in morphology of CaCO3 precipitates was observed, namely, the spherulite morphology (Figures 2f, 5g, and 6a,d,g,j). The SEM micrographs in Figures 5h,i and 6b,c,e,f,h,I,k,l, acquired in the SE and BSE modes, reveal that the spaces between the precipitates were filled with organic matrixes (the polypeptides and gel matrixes) and the surfaces became more rough as the amount of PAsp and PLys added increased. The FTIR spectrum of the spherulites formed with PAsp alone (2 mg/10 mL) showed bands at 713 and 874 cm−1, corresponding to the typical vibrational bands of calcite, bands at 1379 and 1578 cm−1, corresponding to the carboxylate groups of PAsp,35,36 and a weak broadband at around 1072 cm−1 attributed to the gel matrix, suggesting that the spherulites consisted of calcite aggregates with PAsp and a trace amount of the gel matrix (Figure 7a). The FTIR spectrum of PAsp alone also showed peaks at 1400 and 1582 cm−1, corresponding to the carboxylate groups of PAsp. Hence, the red-shifts in the bonds corresponding to the carboxylate groups of PAsp were identified, indicating the presence of PAsp−Ca2+ complexes in the spherulites.19 The XRD pattern of the spherulites also verified that the crystal phase was calcite only (Figure 7b). A XRD pattern and a FTIR spectrum of the precipitates obtained in the presence of a mixture of PLys (4 mg/10 mL) and PAsp (2 mg/10 mL) are shown in Figure 8a,b. The XRD pattern showed diffraction peaks at 29.4, 36.0, and 39.4° and FTIR bands were observed at 713 and 874 cm−1, indicating that the polymorph is calcite.35,37 In addition, the FTIR spectrum showed the characteristic band at 1373 cm−1 attributable to PAsp and the bands at the region of 1650− 1570 cm−1 attributable to PAsp and PLys (Figure 8b). There was also an overlap between the bands of PLys and gel matrix

Figure 8. XRD pattern and FTIR spectra of the spherulites obtained in the presence of a mixture of PLys (4 mg) and PAsp (2 mg): (a) XRD pattern of the spherulites; (b) FTIR spectra of PAsp alone (solid orange line), PLys alone (solid green line), and the spherulites (solid red line); and (c) FTIR spectrum of residue of the spherulite from which the mineral component was removed with HCl.

at around 1060 cm−1. These results suggest the presence of PLys, PAsp, and gel matrix in the spherulites. The highmagnification SEM micrographs acquired in the SE and BSE modes show that the spherulites were composed of interconnected microparticles (Figures 5h,i and 6b,c,e,f,h,I,k,l). The sizes of the microparticles determined from the highmagnification SEM micrographs (Figures 5h,i and 6b,c,e,f,b,c,e,f,h,I,k,l) were as follows: 0.5−3.0 μm (for PAsp 2.0 mg alone), 0.5−2.5 μm (for the mixture of PLys 2 mg and PAsp 1.0 mg), 0.5−2.0 μm (for the mixture of PLys 2 mg and PAsp 2.0 mg), 0.5−1.0 μm (for the mixture of PLys 4 mg and PAsp 1.0 mg), and 0.5−0.7 μm (for the mixture of PLys 4 mg and PAsp 2.0 mg). The sizes of the microparticles were thus manipulated by varying the amounts of PLys and PAsp in the mixture. The resulting crystal polymorph, morphology, and crystal size of the calcite precipitates formed in the presence 16687


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of the −COO− groups from 1400 to 1383 cm−1 due to the copolymerization of PLys and PAsp, that is, the formation of polyion complex assemblies. The FTIR spectrum was similar to that of the polyaspartic acid−lysine copolymer in ref 39. 3.4. Internal Structures of CaCO3 Precipitates. To examine the growth history and internal crystal structure of CaCO3 spherulites, cross-sectional images of the calcite and vaterite spherulites and calcite dendrites were acquired by embedding them in epoxy resin, followed by mechanical polishing and etching of the surface with 0.01 M HCl solution for 2 s. The cross-sectional SEM micrographs (in SE and BSE modes) of the calcite dendrite (Figure 9a−c) and the platelike crystals composing the vaterite spherulites (Figure 9d−f) indicate that organic matrixes (PLys and gel matrixes) are incorporated in the dendrite crystals and vaterite spherulites. The cross-sectional SEM micrographs of the calcite spherulites formed with PAsp alone (Figure 9g,h) and a mixture of PLys and PAsp (Figure 10a,c,i,m) reveal that the calcite spherulites had a hierarchal structure with an inner part (the core) having a loose structure and an outer part composed of rodlike calcite crystals that grew radically from the core.40−42 Some of the cores are partially or completely dissolved during the crystallization process. Unfortunately, for the vaterite spherulites and calcite dendrites formed in the presence of PLys alone, no core was observed (Figure 9a,d). Moreover, comparison of the SE mode cross-sectional SEM micrographs of the calcite spherulites with those acquired in the BSE mode demonstrated that the outer part was composed of a large number of rodlike calcite crystals and organic matrixes (the polypeptides, polyion complex assemblies, and gel matrixes) were present in the calcite spherulites (Figures 9g−i and 10a,b,e,f,i,j,m,n). The high-magnification SEM micrographs acquired in the SE and BSE modes (Figure 10c,d,g,h,k,l,o,p)

Table 2. Resulting Crystal Polymorph, Morphology, and Crystal Size polypeptide

amount added (mg/10 mL)

polymorph

morphology

size (μm)a

rhombohedra dendrite spherulite spherulite spherulite

75−1.8 × 102 60−2.6 × 102 40−1.7 × 102 8−23 23−90

control PLys

0 4

PAsp PLys and PAsp PLys and PAsp PLys and PAsp PLys and PAsp

2 4 and 2

calcite calcite vaterite calcite calcite

4 and 1

calcite

spherulite

40−1.1 × 102

2 and 2

calcite

spherulite

15−80

2 and 1

calcite

spherulite

30−1.0 × 102

a

In rhombohedral morphology, the size is defined as the length along an edge and in spherical morphology, and the size is done as the diameter.

and absence of the polypeptides are listed in Table 2. The calcite spherulites tended to become larger with an increase in the content of PLys and a decrease in the amount of PAsp. To examine the polypeptide matrixes within the spherulite in detail, a residue of the calcite spherulites, which are formed with the mixture of PLys (4 mg) and PAsp (2 mg) with HCl treatment, was prepared. The FTIR spectrum of the residue obtained via the KBr technique showed no crystalline phase of calcite and narrow bands at 1403 cm−1 (−COO−) and 1634 cm−1 (amide I), a broadband at 1068 cm−1 (overlapping between the bands of PLys and gel matrix), and an additional band at 1383 cm−1 (Figure 8c).38,39 We discuss that the additional band at 1383 cm−1 results from the red-shift of some

Figure 9. SE and BSE mode cross-sectional SEM micrographs of precipitates formed in the presence of PLys alone and PAsp alone: (a−f) with PLys alone (4 mg) and (g−i) with PAsp alone (2 mg). The scale bars are 10 μm (a, d, g) and 5 μm (b, c, e, f, h, i). Note that panels, (a, b, d, e, g, and h) are the SEM micrographs acquired in the SE mode and panels, (c, f, and i) are the SEM micrographs in the BSE mode. 16688


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Figure 10. SE and BSE mode cross-sectional SEM micrographs of calcite spherulites formed in the presence of a mixture of PLys and PAsp: (a−d) with PLys (4 mg) and PAsp (2 mg); (e−h) with PLys (4 mg) and PAsp (1 mg); (i−l) with PLys (2 mg) and PAsp (2 mg); and (m−p) with PLys (2 mg) and PAsp (1 mg). Note that panels, (a, c, e, g, i, k, m, and o) are the SEM micrographs acquired in the SE mode and panels, (b, d, f, h, j, l, n, and p) are the SEM micrographs in the BSE mode. The scale bar is 10 μm.

Figure 11. Photograph of calcite spherulite (a) and distribution map of calcite (red area) (b) obtained by micro-FTIR. The spectrum was collected between 4000 and 700 cm−1 at a spectral resolution of 4 cm−1. The distribution map has a pixel size of 6.25 × 6.25 μm2.

also suggest that the organic matrixes in the core were arranged in a mosaic pattern and there was a distinct boundary between the outer part and the core. On the basis of the discussion mentioned already, we judge that the core consists of the organic matrixes composed mostly of polyion assemblies and poorly crystalline calcium carbonate. Micro-FTIR analysis was

carried out to obtain the distribution maps of calcite and the organic matrixes in the spherulites. The outer and inner parts evidently comprised of different components, where the outer part was rich in calcite (Figure 11). Unfortunately, the organic matrixes in the core were not identified due to the poor spectrum from the core. Therefore, we would like to discuss 16689


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Figure 12. Possible sequences for the formation of hollow calcite spherulites. At the initial stage, polyion complex assemblies were formed from the polypeptides (PLys and PAsp) (a−c). The polyion complexes-poorly crystalline calcium carbonate composites develop into the core that acts as a template for nucleation of the calcite crystals (d). In the next stage, the rodlike calcite crystals grow radially from the surface of the core that acts as a substrate for the overgrowth of the outer part (e). In the closed system under double-diffusion conditions, a periodic change in the ionic product (Ca2+)(CO32−) occurs, resulting in repeated crystallization of the calcite crystals (f) and dissolution of the core (g, h).

double-diffusion conditions, a periodic change in the ionic product (Ca2+)(CO32−) occurs by balancing the decrease in the reactant concentration due to crystallization with the supply of the flux of each reactant from the source due to the finite diffusion system, and the ionic product (Ca2+)(CO32−) simultaneously decreases. The supersaturation is given by [(Ca2+)(CO32−)/Ks,x]1/2, where Ks,x is the thermodynamic solubility product of the polymorph x. Therefore, further growth of the rodlike calcite crystals stops below a threshold supersaturation. When the supersaturation reaches an optimum value, crystallization is achieved again and the rodlike calcite crystals are formed on the old crystals (Figure 12f). These crystallization processes occur repeatedly, resulting in the formation of the outer part with a radial growth structure. Hollow spherulites are formed by the dissolution of the unstable core with poorly crystalline calcium carbonate under conditions of the decrease in the ionic product (Ca2+)(CO32−) in the closed gel growth system, as already mentioned (Figure 12g,h).

this matter as the subject of a future study. The core size obtained with the mixtures of PLys and PAsp decreased with the composition as follows: PLys (4 mg) + PAsp (1 mg) > PLys (4 mg) + PAsp (2 mg) > PLys (2 mg) + PAsp (1 mg) > PLys (2 mg) + PAsp (2 mg). The smallest core size was achieved with PAsp alone. The hollow size corresponding to the core size could thus be controlled by changing the relative amount of PLys and PAsp in the mixture. 3.5. Possible Process for Formation of Calcite Hollow Spherulites. On the basis of the SEM observations and the experimental results, we propose a possible process for the formation of the calcite hollow spherulites in the presence of the mixture of PAsp and PLys under double-diffusion conditions in the agar hydrogels, as schematically represented in Figure 12. When PLys and PAsp are added to the agar solution (pH = 8.0) before gelation, some of the polypeptides form a polyion complex assembly with a loose structure (Figure 12a,b). The polyion complex assemblies are surrounded by a shell of excess polyion chain (PAsp) with carboxylate (−COO−) groups. In the next stage, the reactant ions (Ca2+ and CO32− ions) diffuse at the start of the experiment, and some of the unsatisfied carboxylate (−COO−) groups in the polyion complex assemblies then interact with Ca2+ ions in the gel (Figure 12c). When the threshold equivalent concentration of Ca2+ ions and CO32− ions for nucleation is reached in the vicinity of the assemblies, poorly crystalline calcium carbonate crystallizes on the assemblies. Then, the polyion complexes-poorly crystalline calcium carbonate composites develop into the core that acts as a template for nucleation of the calcite crystals (Figure 12d). After this stage, calcite crystallization occurs at multiple nucleation sites on the core, and calcite crystals with the rodlike morphology grow radially from the core in the presence of mobile polypeptides in the gel (Figure 12e). The mobile polypeptides in the gel inhibit the growth of the calcite crystals through their adsorption. In the closed system under

4. CONCLUSIONS The crystallization of calcium carbonate in the presence of PLys and PAsp was evaluated under double-diffusion conditions in agar hydrogels. The crystallization of CaCO3 precipitates was discussed based on the equivalency rule, where CaCO3 nucleation in the presence of PLys and PAsp occurred heterogeneously on the polyion complex assemblies that acted as templates for nucleation. Without the polypeptides, the crystal phase of the precipitates was calcite only and the crystallites adopted a rhombohedral morphology. With PLys alone, the precipitates comprised of calcite as the major component and vaterite as the minor component, where calcite and vaterite occurred as dendrites with a star-like shape and spherulites, respectively. In the presence of PAsp alone and a mixture of PLys and PAsp, the precipitates comprised calcite 16690


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(11) Prieto, M.; Fernádez-Diaz, L.; López-Andrés, S. Spatial and evolutionary aspects of nucleation in diffusing-reacting systems. J. Cryst. Growth 1991, 108, 770−778. (12) Wada, N.; Horiuch, N.; Nishio, M.; Nakamura, M.; Nozaki, K.; Nagai, A.; Hashimoto, K.; Yamashita, K. Crystallization of calcium phosphate in agar hydrogels in the presence of polyacrylic acid under double diffusion conditions. Cryst. Growth Des. 2017, 17, 604−611. (13) Asenath-Smith, E.; Li, H.; Keene, E. C.; She, Z. W.; Estroff, L. A. Crystal growth of calcium carbonate in hydrogels as a model of biomineralization. Adv. Funct. Mater. 2012, 22, 2891−2914. (14) Ajikumar, P. K.; Lakshminarayanan, R.; Valiyaveettil, S. Controlled deposition of thin films of calcium carbonate on natural and synthetic templates. Cryst. Growth Des. 2004, 4, 331−335. (15) Sugawara, A.; Oichi, A.; Suzuki, H.; Shigesato, Y.; Kogure, T.; Kato, T. Assembled structures of nanocrystals in polymer/calcium carbonate thin-film composites formed by the cooperation of chitosan and poly(aspartate). J. Polym. Sci., Part A: Polym. Chem. 2006, 44, 5153−5160. (16) Euliss, L. E.; Bartl, M. H.; Stucky, G. D. Control of calcium carbonate crystallization utilizing amphiphilic block copolypeptides. J. Cryst. Growth 2006, 286, 424−430. (17) Cheng, X.; Varona, P. L.; Olszta, M. J.; Gower, L. B. Biomimetic synthesis of calcite films by a polymer-induced liquidprecursor (PILP) process: 1. Influence and incorporation of magnesium. J. Cryst. Growth 2007, 307, 395−404. (18) Sancho-Tomás, M.; Fermani, S.; Durán-Olivencia, M. A.; Otálora, F.; Gómez-Morales, J.; Falini, G.; García-Ruiz, J. M. Influence of charged polypeptides on nucleation and growth of CaCO3 evaluated by counter diffusion experiments. Cryst. Growth Des. 2013, 13, 3884−3891. (19) Wada, N.; Horiuch, N.; Nakamura, M.; Nozaki, K.; Hiyama, T.; Nagai, A.; Yamashita, K. Cooperative effects of polarization and polyaspartic acid on formation of calcium carbonate films with a multiple phase structure on oriented calcite substrates. J. Cryst. Growth 2014, 402, 179−186. (20) Yao, Y.; Dong, W.; Zhu, S.; Yu, X.; Yan, D. Novel morphology of calcium carbonate controlled by poly(L-lysine). Langmuir 2009, 25, 13238−13243. (21) Schenk, A. S.; Cantaert, B.; Kim, Y. Y.; Li, Y.; Read, E. S.; Semsarilar, M.; Armes, S. P.; Meldrum, F. C. Systematic study of the effects of polyamines on calcium carbonate precipitation. Chem. Mater. 2014, 26, 2703−2711. (22) Wada, N.; Horiuch, N.; Nakamura, M.; Nozaki, K.; Nagai, A.; Yamashita, K. Calcite crystallization on polarized single calcite crystal substrates in the presence of polylysine. Cryst. Growth Des. 2018, 18, 872−879. (23) Pučar, Z.; Pokrić, B.; Graovac, A. Precipitation in gels under conditions of double diffusion: Critical concentrations of the precipitating components. Anal. Chem. 1974, 46, 403−409. (24) Ž ivković, T.; Pokrić, B.; Pučar, Z. Stoichiometry of precipitation under conditions of double diffusion. J. Chem. Soc., Faraday Trans. 1 1974, 70, 1991−1998. (25) Wada, N.; Yamashita, K.; Umegaki, T. Effects of carboxylic acids on calcite formation in the presence of Mg2+ ions. J. Colloid Interface Sci. 1999, 212, 357−364. (26) Wada, N.; Yamashita, K.; Umegaki, T. Effects of divalent cations upon nucleation, growth and transformation of calcium carbonate polymorphs under conditions of double diffusion. J. Cryst. Growth 1995, 148, 297−304. (27) Burke, S. E.; Barrett, C. J. pH-responsive properties of multilayered poly(L-lysine)/hyaluronic acid surfaces. Biomacromolecules 2003, 4, 1773−1783. (28) Wu, Y. T.; Grant, C. Effect of chelation chemistry of sodium polyaspartate on the dissolution of calcite. Langmuir 2002, 18, 6813− 6820. (29) Insua, I.; Wilkinson, A.; Fernandez-Trillo, F. Polyion complex (PIC) particles: Preparation and biomedical applications. Eur. Polym. J. 2016, 81, 198−215.

spherulites only. The spherulites adopted a hierarchal structure with an inner part (the core) having a loose structure and the outer part composed of rodlike calcite crystals that grew radically from the core. Finally, complete dissolution of the cores provided spherulites with a hollow structure. It is speculated that the core consists of polyion complex assemblies and poorly crystalline CaCO3. The synergistic effects of PAsp and PLys were found to be operative in calcite crystallization, where changing the relative amount of PLys and PAsp in the mixture had the effects of (a) regulating the sizes of the microparticles composed of the calcite crystals and (b) controlling the hollow size of the calcite spherulites and simultaneously the calcite spherulite sizes. A possible process of the formation of the calcite hollow spherulites in the presence of the mixture of PAsp and PLys under doublediffusion conditions in agar hydrogels was proposed. This study may provide new insights into and important information for understanding the effects of organic molecules on biomineralization and for biomimetic syntheses of biomaterials.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel/Fax: +81-03-5280-8015. ORCID

Norio Wada: 0000-0003-3883-197X Naohiro Horiuchi: 0000-0002-7804-2337 Notes

The authors declare no competing financial interest.

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REFERENCES

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Controlled Crystallization of Calcium Carbonate via Cooperation of

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